Release of retinal growth hormone in the chick embryo: local regulation?

The neural retina is an extrapituitary site of growth hormone (GH) production and an autocrine or paracrine site of retinal GH action. Retinal GH is released from retinal tissue and may be secreted into the vitreous. Ontogenetic changes in the abundance of retinal GH during embryogenesis indicate that the amount of GH released may be regulated. The presence of pituitary GH secretagogues (GH-releasing hormone, GHRH; thyrotropin-releasing hormone, TRH; and ghrelin) and pituitary GH inhibitors (somatostatin, SRIF and insulin-like growth factor, IGF-1) within the neural retina may indicate the involvement of these factors in retinal GH release. This possibility is supported by the finding that GHRH is colocalized with GH in chick retinal ganglion cells (RGCs) and in immortalized cells (QNRD) derived from quail neuroretinal cells and by the induction of GH mRNA in incubated QNRD cells. In summary, these results provide evidence for the autocrine or paracrine regulation of retinal GH release in the ganglion cells of the embryonic chick retina.

Growth hormone promotes the survival of retinal cells in vivo.

Growth hormone (GH) is synthesized and present in the developing chick retina, where it may have local actions in retinal cell differentiation similar to those of conventional growth factors. We have previously shown that retinal GH has neuroprotective effects in retinal ganglion cells. In this paper, we extend our earlier functional studies by examining the in vivo effects of a GH siRNA (NR-cGH-1) after microinjection into the eye cup of the developing chick embryo in ovo. We show that intra-vitreous cGH siRNA lowers both GH mRNA and insulin-like growth factor-1 (IGF-1) mRNA levels in the retina in vivo, and concomitantly elevates the numbers of apoptotic cells in the retina. These effects are apparent 6h after treatment, and persist for at least 24h. The apoptotic cells induced by GH withdrawal were primarily located close to the optic fissure of the developing eye, and were distributed in clusters, suggesting that there are sub-populations of retinal cells that are particularly susceptible to apoptotic stimuli. These results support our view that a GH/IGF-1 axis in retinal cells regulates retinal cell survival in vivo.

Growth hormone expression and neuroprotective activity in a quail neural retina cell line.

We have previously shown that growth hormone (GH) is produced within cells of the chick embryo retina where it appears to act as an autocrine/paracrine anti-apoptotic factor in the regulation of programmed cell death during retinal development. These investigations were carried out on cultured chick embryo retinal ganglion cells (RGCs) as well as on the chick embryo retina in ovo, using GH protein knock-down by immunoneutralization. We have now investigated the putative neuroprotective actions of GH using a quail embryo neural retina cell line (QNR/D) treated with GH siRNA to silence the local synthesis of GH. We now show that knock-down of GH by gene silencing in cells of this cultured embryonic neural retina cell line, using NR-cGH-1 siRNA, correlates with the increased appearance in the cultures of cells with apoptotic nuclear morphology. This result is consistent with our previous results using protein knock-down by immunoneutralization. We thus validate, using different technology and a different culture system, our contention that GH, produced locally by cells of the neural retina acts in an autocrine or paracrine manner to regulate cell survival in the retina.

Endogenous growth hormone in human retinal ganglion cells correlates with cell survival.

PURPOSE: Locally synthesized growth hormone (GH) may act as a survival factor in several tissues. Experimental studies with chick retinal ganglion cells (RGCs) suggest that GH, synthesized within the developing retina, may have autocrine or paracrine roles in the regulation of the waves of cell death characteristic of RGC differentiation. There is also evidence that GH may have a similar neuroprotective function in the rat retina, however, there is no information concerning such a role in the human retina. In this paper we extended our earlier work by determining whether the local expression of retinal GH correlates with RGC apoptosis in human retinas. METHODS: In the absence of experimental approaches to survival factor function in the human retina, we have used a correlative immunocytochemical technique to determine how the expression of GH relates to cell death in RGCs. We used sections of human retinas, taken postmortem, that we double-labeled for GH and apoptotic cell death using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). RESULTS: We found that approximately 35% of cells in the ganglion cell layer (GCL) were both GH positive and GH receptor (GHR) positive, and that GH colocalized with GHR in these cells. However, none of the apoptotic cells in the GCL were GH immunoreactive. Labeling of sections with the RGC marker, synuclein, indicated that at least 95% of the cells in the GCL were RGCs, leading us to conclude that the majority of the cells that we have examined in the GCL are RGCs. CONCLUSIONS: The results are consistent with the earlier proposal, based on in vivo and in vitro experimental chick embryo studies, that GH promotes survival in adult human RGCs.

Growth hormone-induced neuroprotection in the neural retina during chick embryogenesis.

Recent studies have established that retinal ganglion cells (RGCs) of the neural retina are extrapituitary sites of growth hormone (GH) production and action in early chick embryos prior to the ontogeny of pituitary somatotrophs. The presence of GH in axons of the RGCs is, however, restricted to the period when the retinofugal neurons project to and synapse with visual centers within the brain, suggesting roles for GH in axonal growth or guidance. Autocrine and/or paracrine actions of retinal GH are also neuroprotective for RGCs during developmental waves of apoptosis that characterize RGC differentiation. The anti-apoptotic actions of retinal GH use signaling mechanisms that are common to other established neurotrophins (e.g., brain-derived growth factor, insulin-like growth factor-1, transforming growth factor beta-1). Retinal GH is therefore a novel neurotrophin in the visual system during chick embryogenesis.

Vitreous growth hormone and visual dysfunction.

Growth factors have been found in vitreous fluid, in which they regulate retinal function and provide markers of ocular dysfunction. Since growth hormone (GH) has recently been discovered in the vitreous of human eyes, the possibility that vitreal GH concentrations might differ in different ocular disease states was assessed. GH-immunoreactivity in the vitreous of cadaver controls and in the vitreous of patients with ocular dysfunction was quantified by ELISA. In non-diabetics, vitreous GH concentrations were comparable in patients with subretinal hemorrhage (SH), vitreous hemorrhage (VH), vitreous debris (VD), retinal detachment (RD), central retinal vein occlusion (CRVO), macular hole (MH), dislocated crystalline lens (DCL) or epiretinal membrane (ERM). The GH concentration, corrected for protein content, in the vitreous of diabetic patients was, however, lower than that in cadaver controls with no history of ocular disease and lower than that in non-diabetic patients with ocular dysfunction. Vitreous GH concentrations in diabetic patients with proliferative diabetic retinopathy (PDR) did not differ from those without PDR. The presence of GH in the human vitreous suggests that vitreous GH may have roles in normal ocular function and be involved in the pathogenesis of ocular disease. Low GH concentrations in the vitreous of diabetic patients may correlate with retinal neurodegeneration and may provide a marker to follow its progression.

Growth hormone is present in the human retina and vitreous fluid.

Growth factors have been found in vitreous fluid, in which they regulate retinal function and provide markers of ocular dysfunction. Since growth hormone (GH) has recently been discovered in the eyes of rodents and embryonic chicks and found to be neuroprotective for retinal ganglion cells, the possible presence of GH in the human retina and vitreous fluid has been assessed. GH-immunoreactivity in the retina and vitreous fluid of cadavers and in the vitreous fluid of patients with ocular dysfunction was determined by Western blotting. GH-immunoreactivity, identical in size (22kDa) to recombinant pituitary GH was found in proteins extracted from the retina and in the vitreous fluid of patients with ocular disease (proliferative diabetic retinopathy, epiretinal membrane and vascular hemorrhage) and individuals with no history of ocular disease. GH-immunoreactivity was also detected in large, discrete cells in the retinal ganglion cell layer, in which GH staining was mainly within the nuclear compartment. The novel presence of GH in the human retina and vitreous fluid suggests GH may have roles in visual function and be involved in the pathogenesis of ocular disease.

Signaling mechanisms mediating local GH action in the neural retina of the chick embryo.

Growth hormone (GH) is found in the retina and vitreous of the chick embryo, where it appears to act as a growth and differentiation factor, having neuroprotective effects on retinal ganglion cells (RGCs). Here, we review the molecular mechanisms of the anti-apoptotic effect of GH in chick RGCs. GH treatment of RGCs reduces Akt levels, while raising Akt-phos levels, consistent with a role for Akt signaling pathways in the GH neuroprotective action. The induction of apoptosis by immunoneutralization with GH antiserum is accompanied by an increase in caspase-3 and caspase-9 activation, and also PARP-1 cleavage. Calpain activation also appears to be a major caspase-independent pathway to PARP-1 cleavage and apoptosis in these cells, supporting the view that caspase and calpain inhibitors are major neuroprotective agents for RGCs, and that pathways that activate both caspases and calpains are important for the anti-apoptotic actions of GH in these cells. These pathways involve the activation of cytosolic tyrosine kinases (Trks) and extracellular-signal-related kinases (ERKs). Occupation of the GH receptor by GH involves downstream intracellular Trk pathways. The Akt and Trk pathways appear to converge on the activation of cAMP response element binding protein (CREB), which is able to initiate transcription of pro- or anti-apoptotic genes. These results indicate that the action of GH in the neuroprotection of embryonic RGCs involves pathways common to with other neurotrophins, and that GH can be considered to be a growth and differentiation factor in the development of the embryonic retina. We have also investigated the relationship between the overlapping anti-apoptotic effects of GH and insulin-like growth factor-1 (IGF-1), two functionally closely related factors. We find that simultaneous immunoneutralization of GH and IGF-1 does not increase the level of apoptosis in the cultures above that achieved by immunoneutralization of GH alone. We therefore conclude that the neuroprotective actions of GH in the developing retina are likely mediated in large part through the action of IGF-1.

Peptide hormones as developmental growth and differentiation factors.

Peptide hormones, usually considered to be endocrine factors responsible for communication between tissues remotely located from each other, are increasingly being found to be synthesized in developing tissues, where they act locally. Several hormones are now known to be produced in developing tissues that are unrelated to the endocrine gland of origin in the adult. These hormones are synthesized locally, and are active as differentiation and survival factors, before the developing adult endocrine tissue becomes functional. There is increasing evidence for paracrine and/or autocrine actions for these factors during development, thus, placing them among the conventional growth and differentiation factors. We review the evidence for the view that thyroid hormones, growth hormone, prolactin, insulin, and parathyroid hormone-related protein are developmental growth and differentiation factors.

Growth hormone-mediated survival of embryonic retinal ganglion cells: signaling mechanisms.

Growth hormone (GH) is found in the developing eye, where it is synthesized by retinal ganglion cells (RGCs). In this location, GH variants appear to have an autocrine or paracrine anti-apoptotic neuroprotective role, and may contribute to the regulation of the developmental waves of apoptosis that characterize RGC differentiation. Here, we investigate the intracellular signaling pathways that are activated by GH as a neuroprotective agent in cultured chick embryo RGCs. We show that GH treatment reduces the cleavage of caspase-9, and that an inhibitor of caspase-9 cleavage can abrogate the pro-apoptotic effect of GH immunoneutralization. These findings complement previous results implicating caspase-3 in GH action on these cells. We had also previously shown that Akt pathways are involved in the neuroprotection of RGCs by GH. We now extend those findings to show that these pathways involve the activation of cytosolic tyrosine kinases (Trks) and extracellular-signal-related kinases (ERKs). Therefore, although the GH receptor, unlike other neurotrophin receptors, is not itself a receptor tyrosine kinase (receptor Trk), occupation of the receptor by GH involves downstream intracellular Trk pathways. Finally, we show that the Akt and Trk pathways converge on the activation of cAMP response element binding protein (CREB) which is able to initiate transcription of pro- or anti-apoptotic genes. These results indicate that the action of GH in the neuroprotection of embryonic RGCs involves pathways that are common to other neurotrophins, and that GH can be considered to be an authentic growth and differentiation factor in the development of the embryonic retina.

Growth hormone and developmental ocular function: clinical and basic studies.

While growth hormone (GH) is obligatory for postnatal growth, its possible involvement in ocular development and vision is poorly understood. The eye is, however, a target site for GH action and GH production and GH may have endocrine, autocrine and/or paracrine roles in ocular development. The importance of GH in ocular development is demonstrated by the ocular abnormalities that can occur in patients with pituitary GH excess or GH deficiency. Clinical and basic studies supporting roles for GH in ocular development are the focus of this brief review.

Growth hormone in the visual system: comparative endocrinology.

Growth hormone (GH) is rarely considered to be involved in ocular development or vision or to be present in the visual system. Basic and clinical studies nevertheless support roles for GH in the ocular function of most vertebrate groups and for its extrapituitary production in ocular tissues. The comparative endocrinology of endocrine, autocrine or paracrine GH in the visual system of vertebrates is the focus of this brief review.

Growth hormone and cell survival in the neural retina: caspase dependence and independence.

Growth hormone has recently been shown to be expressed in the retinal ganglion cells of embryonic chicks, in which it induces cell survival during neurogenesis. The mechanism of this action has been examined in neural retina explants from 6-day-old and 8-day-old embryos that were incubated for 48 h in 10 M growth hormone, to reduce the number of spontaneous apoptotic cells. This anti-apoptotic action was accompanied by a reduction in caspase-3 expression and, at embryonic day 8, by reduced expression of apoptosis inducing factor-1, which is caspase independent. These actions were specific, as other genes involved in apoptotic signaling (bcl-2, bcl-x, bid and inhibitor of apoptosis protein-1) were unaffected. These results therefore demonstrate caspase-dependent and caspase-independent pathways in growth hormone-induced retinal cell survival.

Retinal ganglion cell survival in development: mechanisms of retinal growth hormone action.

Several variants of growth hormone (GH) are found in the retina and vitreous of the chick embryo, where they appear to act as cell survival factors, having neuroprotective effects on retinal ganglion cells (RGCs). Here, we investigate the molecular mechanisms of the anti-apoptotic effect of GH in cultured RGCs. GH treatment increased Akt phosphorylation in these cells, which is an anti-apoptotic event. Whereas unphosphorylated Akt was detected in both nucleus and cytoplasm of RGCs by immunocytochemistry, the phosphorylated form of Akt (Akt-phos) was located primarily in the cytoplasm of both normal and apoptotic cells, although levels were markedly lower in the latter. It was found that GH treatment of RGCs reduced Akt levels, while concomitantly raising Akt-phos levels, consistent with a role for Akt signaling pathways in GH neuroprotective action. This was substantiated using Wortmannin, which, like GH antiserum, inhibited Akt phosphorylation and initiated apoptosis. The addition of Wortmannin to RGC cultures simultaneously with GH significantly reduced the anti-apoptotic effect of GH. The induction of apoptosis by GH antiserum was clearly accompanied by an increase in caspase-3 activation and PARP-1 cleavage, both of which were significantly reduced in the presence of the broad spectrum caspase inhibitor, Q-VD-OPh, which itself had a dramatic neuroprotective effect on cultured RGCs. Calpain activation appeared to be a major caspase-independent pathway to PARP-1 cleavage and apoptosis in these cells. Calpain inhibitor III (MDL 28170) was able to reduce PARP-1 cleavage and abrogate the apoptogenic effect of GH antiserum. The results support the view that caspase and calpain inhibitors are major neuroprotective agents for RGCs, and that pathways that activate both caspases and calpains are important for the anti-apoptotic actions of GH in these cells.

Retinal growth hormone in perinatal and adult rats.

Growth hormone (GH) mRNA and protein have recently been localized in the neural retina of embryonic chicks, in which exogenous GH promotes cell survival. GH is also expressed in the rat CNS, in which it has neuroprotective roles, although its presence in the rat neural retina is unknown and is the focus of the present study. GH immunoreactivity, to a 22-kDa protein, was present in extracts of fetal (embryonic day [ED]17) eyes and in extracts from the neural retinas of newborn pups, comparable to GH immunoreactivity in pituitary extracts. The GH immunoreactivity in the neural retina was widespread but was most intense in large rounded cells in the retinal ganglion cell (RGC) layer and in the optic fiber layer derived from the axons of the RGCs. A 693-bp cDNA was also generated by the RT-PCR of RNA extracted from the eyes of ED17 rats and from the neural retinas and eyes of newborn rats, when amplified in the presence of oligonucleotide primers for the rat GH cDNA. Expression of the GH gene in the neural retina was also shown by specific in situ hybridization of an antisense GH riboprobe to cells in the neural retina, particularly those in the RGC layers of fetal and adult rat eyes. These results demonstrate GH expression in the neural retinas of fetal, newborn, and adult rats, in which retinal GH might have neuroprotective roles.

Retinal growth hormone is an anti-apoptotic factor in embryonic retinal ganglion cell differentiation.

Cells of the neural retina in the chick embryo undergo several waves of apoptosis during development, including peaks at approximately embryonic day (ED) 7 and 12. Prominent among the cells involved in these phases of cell death are the retinal ganglion cells (RGCs). We have previously shown that growth hormone (GH) is expressed in the neural retina, and particularly, in the RGCs. Here we study the ability of GH to rescue retinal cells from apoptosis, both in vitro and in vivo. When retinas from embryos at ED 6-8 are explanted on collagen gels, the application of recombinant GH, at 10(-6)m, significantly reduced the incidence of apoptotic cells in the cultures as judged by terminal deoxynucleotide transferase-mediated dUTP-biotin nick end labelling (TUNEL). GH was delivered to neural retinas in ovo, by microinjection into the eye cup at ED 2. When these embryos were examined at ED 6-8, no reduction in cell death was observed below the normal low control levels. However, when antibodies to GH were microinjected, the incidence of cell death increased significantly at ED 6, providing evidence that in vivo immunoneutralization of endogenous GH results in triggering of apoptotic signaling pathways. Evidence that RGCs are a particular target of this neuroprotective effect of GH was provided by examination of cultures enriched for RGCs by immunopanning. In serum-free culture, RGCs, identified by anti-Islet 1 immunolabelling, were found to be susceptible to the effect of GH immunoneutralization, which approximately quadrupled the incidence of apoptosis in the cultures. We propose that GH is a naturally occurring autocrine and/or paracrine neuroprotective agent in the developing retina which is involved in the regulation of retinal cell numbers during early embryogenesis.

Growth hormone as an early embryonic growth and differentiation factor.

In this review we consider the evidence that growth hormone (GH) acts in the embryo as a local growth, differentiation, and cell survival factor. Because both GH and its receptors are present in the early embryo before the functional differentiation of pituitary somatotrophs and before the establishment of a functioning circulatory system, the conditions are such that GH may be a member of the large battery of autocrine/paracrine growth factors that control embryonic development. It has been clearly established that GH is able to exert direct effects, independent of insulin-like growth factor-I (IGF-I), on the differentiation, proliferation, and survival of cells in a wide variety of tissues in the embryo, fetus, and adult. The signaling pathways behind these effects of GH are now beginning to be determined, establishing early extrapituitary GH as a bona fide developmental growth factor.

Growth hormone localization in the neural retina and retinal pigmented epithelium of embryonic chicks.

It is well-established that growth hormone (GH) is present in the brain, spinal cord, and peripheral nerves of embryonic chicks, prior to the differentiation of pituitary somatotrophs, but its presence and distribution in retinal tissues is controversial. The possible presence of GH and GH mRNA in retinal tissues of early embryos has therefore been further evaluated. A 466-bp fragment of the pituitary GH cDNA, derived from a portion of exon 3 and spanning exons 4 and 5, was amplified by RT-PCR from reverse-transcribed mRNA from the pituitary glands of juvenile chicks and from the whole eye, neural retina, and retinal pigmented epithelium (RPE) of embryonic-day (ED) 9 chick embryos. In ED 9 embryos, GH immunoreactivity was demonstrated in the choroid and neural retina, in which it was particularly abundant in a layer of cells with the location and morphological appearance of retinal ganglion cells. GH immunoreactivity was also present in tissue sections of the RPE that were bleached to remove the melanin pigment. The intense GH staining in the RPE of ED 9 embryos was also revealed using a fluorescein-labeled GH antibody and confocal microscopy. At the ultrastructural level, GH detected by immunogold electron microscopy was present in the cytoplasm of RPE and neural retinal cells of ED 9 embryos. Although not associated with secretory granules, GH in the RPE was particularly associated with the membranes of the melanin granules. These results demonstrate that the neural retina and RPE are extrapituitary sites of GH production in early chick embryos, prior to the differentiation of the pituitary gland.

Opticin binds retinal growth hormone in the embryonic vitreous.

PURPOSE: Opticin is a small leucine-rich repeat proteoglycan that is abundant in several ocular tissues, including the vitreous. Like other proteoglycans, opticin may have the ability to bind and regulate the release of growth factors. Previous work has shown that isoforms of growth hormone (GH) are present in the embryonic retina, where they may act as a growth factor. The current study was conducted to investigate the possibility that opticin binds retinal GH in the vitreous of the chick embryo. METHODS: The vitreous and retina of embryonic day-8 chicks were examined for the presence of opticin and GH, by Western immunoblot analysis, coimmunoprecipitation, and immunocytochemistry. RESULTS: Opticin associated with GH in the embryonic vitreous to produce a 60- to 62-kDa complex. Opticin and GH were also colocalized in the retina in retinal ganglion cells. CONCLUSIONS: The binding of retinal GH by opticin in the vitreous suggests that GH, secreted by the retinal ganglion cells, may be sequestered and concentrated in the vitreous and could act there as a paracrine differentiation factor in ocular development. During development, opticin could therefore regulate growth factor-like actions of retinal GH, both in the vitreous and the retinal ganglion cells. The physiological roles of GH in this location remain to be determined, but may include the regulation of cell proliferation and cell death.

Retroviral overexpression of bcl-2 in the embryonic chick lens influences denucleation in differentiating lens fiber cells.

During the course of their differentiation, embryonic lens fibers undergo loss of their cytoplasmic organelles and nuclei. The denucleation process bears similarities to the nuclear breakdown that occurs during apoptosis. This has given rise to the hypothesis that this denucleation is analogous to apoptosis, but without the plasma membrane changes characteristic of apoptotic cell death. Previous work has shown that several members of the apoptotic cascade are active during denucleation. Here, we have overexpressed the anti-apoptotic molecule bcl-2 in developing lenses of the 8-day-old chick embryo in ovo, using the replication-competent retrovirus RCAS. We find that lenses overexpressing bcl-2 show varying degrees of distortion in comparison with untreated and negative insert controls, including a more spherical shape and disorganized fiber cells. All overexpressing lenses showed significantly higher numbers of smaller nuclei in the lens core, where denucleation begins. There was no change in cell size or pattern of proliferation. These in vivo results were confirmed in vitro using lens epithelial cell cultures, which differentiate into lentoids. The lentoids in treated cultures showed the same effect on nuclear number and size. We further found that in lenses overexpressing bcl-2 there was a reduction in the activation of caspase-9 and the cleavage of the caspase substrate DFF45, and, in the lens core, a failure of the nuclear chromatin to condense. These results provide strong support for the view that embryonic lens fiber cell denucleation is analogous to the nuclear degradation that occurs during apoptosis, and that similar control pathways are involved in both these phenomena.